1. Field of the Invention
The present invention is related to a bacterium for producing polyhydroxyalkanoate in which the gene coding for polyhydroxyalkanoate depolymerase is disrupted and to a method for producing polyhydroxyalkanoate using the same. Also related is a gene targeting vector to disrupt the gene coding for the polyhydroxyalkanoate depolymerase of the bacterium for producing polyhydroxyalkanoate and a method for disrupting the same.
2. Related Background Art
Until now, it has been reported that many microbes produce and accumulate in the body poly-3-hydroxy butyrate (PHB) or other poly-3-hydroxyalkanoate (PHA) (“Biodegradable plastic handbook”, edited by the biodegradable plastic study group, NTS Inc. P178-197 (1995)). These polymers, like conventional plastics, can be used for producing various products by melt processing and the like. Furthermore, these polymers have an advantage of being completely degraded by microbes in the nature and do not cause pollution by remaining in the natural environment, unlike many conventional synthetic polymers, because they are biodegradable. They are also superior in biocompatibility, and would be expected to have applications as soft material for medical use and the like.
Recently in particular, it is expected that unusual PHA in which substituents other than alkyl group are introduced in the side chain would be very useful considering expanding application of microbially produced PHA, for example an application as a functional polymer. Examples of such substituents include groups containing an aromatic ring (phenyl group, phenoxy group, benzoyl group and the like), unsaturated hydrocarbons, ester group, aryl group, cyano group, halogenated hydrocarbons, epoxides, thioethers and the like.
It has been known that microbially produced PHA can have various compositions and structures depending upon the relevant microbial species, the composition of the medium, the culture condition and the like. Various researches have been carried out on such PHA producing microbes, and the biosynthetic pathway of PHA has been relatively well investigated.
Up until now, polyhydroxyalkanoate synthases are classified into three classes by substrate specificity and subunit composition.
Polyhydroxyalkanoate synthases belonging to “the first class” are found in Ralstonia eutropha, Aeromonas punctata and the like which use, as substrate, thioesters of short-chain-length 3-hydroxyalkanoates with C3-C5 carbons with a coenzyme CoA.
Polyhydroxyalkanoate synthases belonging to “the second class” are found in Pseudomonas oleovolans and Pseudomonas aeruginosa which use, as substrate, thioesters of various “unusual” 3-hydroxyalkanoates as well as medium-chain-length 3-hydroxyalkanoates with C6-C14 carbons with the coenzyme CoA.
In both the first and second classes, polyhydroxyalkanoate synthases are composed of a single subunit of molecular weight 61-73 kDa.
Polyhydroxyalkanoate synthases belonging to “the third class” are found in Allochromatium vinosum and Ectothiorhodospira shaposhnikovii and composed of two different subunits of about 40 kDA. They have a substrate specificity similar to that of the first class polyhydroxyalkanoate syntheses and use thioesters of short-chain-length 3-hydroxyalkanoates with 3-5 carbons with the coenzyme CoA.
On the other hand, there remains much to be done for the degradation pathway of PHA. The Journal of Biological Chemistry 266, 2191 (1991) has disclosed that by treating Pseudomonas oleovolans containing the polyhydroxyalkanoate synthase with the substrate specificity of the “second class” with a chemical mutagen, a mutant strain was obtained which were not able to degrade intracellular PHA and that using this mutant, a gene which complements PHA degrading activity was cloned. According to this paper, the PHA synthase gene (phaC1, phaC2) and the PHA depolymerase (phaZ) gene formed a cluster and assumed an arrangement of phaC1-phaZ-phaC2. Since then, it has been discovered that in other bacteria such as Pseudomonas aeruginosa, Pseudomonas sp. 61-3, Pseudomonas resinovolans, Pseudomonas putida, Pseudomonas mendocina and the like, which contains polyhydroxyalkanoate synthase with the substrate specificity of the second class, the genes are arranged as phaC1-phaZ-phaC2.
Although mutants, in which the PHA depolymerase gene do not function, have been isolated in the process of these studies, the relationship between the PHA depolymerase activity and the amount of PHA accumulated in the cells is not known.
On the other hand, in Japanese Patent Application Laid-Open No. H11-113574, what is shown is depolymerase of poly-3-hydroxy butyrate (PHB), which is isolated from Alcaligenes eutrophas containing polyhydroxyalkanoate synthase that belongs to “the first class”. Further, in Japanese Patent Application Laid-Open No. H9-191887, what is shown is depolymerase of poly-3-hydroxy butyrate (PHB), which is isolated from Alcaligenes faecalis containing polyhydroxyalkanoate synthase that belongs to “the first class”. Still further, in Japanese Patent Application Laid-Open No. H6-086681, what is shown is depolymerase of poly-3-hydroxy butyrate (PHB), which is isolated from Zoogolea ramigera containing polyhydroxyalkanoate synthase that belongs to “the first class”.
Patent Document 1: Japanese Patent Application Laid-Open No. H11-113574
Patent Document 2: Japanese Patent Application Laid-Open No. H9-191887
Patent Document 3: Japanese Patent Application Laid-Open No. H6-086681
Non-patent Document 1: “Biodegradable plastic handbook”, edited by the biodegradable plastic study group, NTS Inc. P178-197 (1995)
Non-patent Document 2: The Journal of Biological Chemistry 266, 2191 (1991)